Parkinson's disease (PD) is characterized by progressive and selective loss of dopaminergic neurons in substantial nigra pars compacta. Although the etiology remains unknown, there is considerable evidence that supports the view that programmed cell death (PCD) contributes, at least in part, to the degeneration of dopaminergic neurons in PD. Recent studies have identified a group of terminal caspases, particularly caspase-3/-7, as the central executive molecules in neuronal PCD. Caspase-3 actively participates in dopaminergic neuronal cell death in response to PD-relevant insults, although how caspase-3 is activated in this process is largely elusive. Furthermore, caspase-3 and other terminal caspases may not be the only executioners of neuronal PCD. A novel pro-apoptotic molecule, designated as AIF (apoptosis-inducing factor), has now been identified. AIF, which is activated and released from the mitochondria upon receiving death signals, and potently promotes high-molecular-weight DNA fragmentation and nuclear apoptosis. Bcl-2 family proteins are important PCD regulators and have been implicated in dopaminergic neuronal cell death. The pro-apoptosis Bcl-2 family member Bax is a well-characterized cell death effector, which, upon activation, targets mitochondria and triggers the cytochrome c-dependent intrinsic pathway. However, whether Bax activates AIF and the mechanism by which Bax is activated in dopaminergic neuronal apoptosis is unknown. We have now obtained exciting preliminary data which suggest that 1) caspase-3 activation in dopaminergic neurons is dependent on the intrinsic pathway; 2) both AIF-dependent and caspase-dependent mechanisms may contribute independently and synergistically to the final execution of dopaminergic neuronal apoptosis; 3) Bax is a direct mediator of AIF release in neurons, and the activation of Bax during dopaminergic cell death appears to be p53-dependent; and 4) Bak may be an important cofactor that enhances the pro-apoptotic effect of Bax at the mitochondria. Therefore, the objective of this project is to determine the role of caspase-3-dependent and AIF-dependent death execution pathways in dopaminergic neuronal cell death following PD-relevant insults and to determine the role of Bax in triggering the activation of these two pathways. The proposed studies will be performed using complementary in vitro and in vivo model systems, and will take advantage of our recent cloning of novel dominant-negative inhibitory mutants of caspase-9, Apaf-1 and AIF, and the availability of Bax-deficient mice. The following specific aims will be addressed: 1. Test the hypothesis that the caspase-9/Apaf-1 intrinsic pathway plays a central role in mediating terminal caspase activation and dopaminergic neuronal cell death following PD-relevant insults. 2. Test the hypotheses that AIF release is an important mechanism underlying caspase-independent dopaminergic cell death, and that AIF-dependent and caspase-3-dependent pathways work synergistically in the execution of cell death. 3. Test the hypotheses that Bax is a direct mediator for AIF release, and Bax contributes to dopaminergic cell death by activating both AIF-dependent and caspase-dependent pathways.